Many important cellular processes are mediated by RNA molecules, and many of the pathways for regulating gene expression involve specific protein - RNA interactions. Virtually all cellular RNAs interact with proteins, and it is not known how proteins recognize specific RNA sites or how they might promote the specific functions of an RNA. The initial goal of the work in this project was to define a few representative, well-behaved systems for which it is feasible, in the long term, to understand 1) how a protein recognizes and modifies the RNA structure, and 2) how the protein - RNA complex accomplishes a specific biological function. Two sets of interactions which illustrate very different aspects of protein RNA recognition, have been defined. In one, ribosomal protein S4 organizes a large and complex region of the 16S rRNA to initiate ribosome assembly; the same protein also binds to its own mRNA as a translational repressor. In the other system, a small, highly conserved rRNA fragment binds the ribosomal protein L11 and a family of peptide antibiotics; both protein and antibiotic binding have specific effects on the GTPase activity of intact ribosomes. The way in which these ligands perturb the RNA structure is thus relevant to ribosome function. By measuring protein or antibiotic affinity for RNAs containing deletions and base changes, the RNA sequences and secondary structures necessary for protein recognition and biological function are being systematically determined. Selection experiments in which about 10(12) RNA sequence variants can be screened for protein recognition are being used to detect additional secondary and tertiary structures. In addition, there is evidence for each of the RNAs under study that the protein perturbs transitions between alternate RNA conformations. The thermodynamics of conformational transitions in these RNAs will be examined, and the effects of protein or antibiotic binding on the transitions determined. 1H and 31P NMR experiments will be carried out on one of the smaller RNAs to detect and locate tertiary structure. The protein binding this RNA, L11, is being overexpressed from a thermophile for NMR studies of its folding. Although these studies will be carried out using fragments of ribosomal or messenger RNA, in each case the physical properties of the fragment RNA - protein complex can be related to the in vivo function of the intact ribosome or messenger. These studies will ultimately be essential for understanding the mechanism of translational repression and the role of proteins in ribosomes, and will also contribute to our general knowledge of strategies used by protein - RNA complexes to carry out specific biological tasks.